The present invention relates to compositions and methods for assaying analytes, preferably, small molecule analytes. More particularly, assay methods that employ, in place of antibodies, modified enzymes that retain binding affinity or have enhanced binding affinity, but that have attenuated catalytic activity, are provided. The modified enzymes are also provided.
Methods for assaying analytes have wide applications. Many analytes including small molecule analytes are essential components and/or participants of biological systems and processes. Methods for assaying these analytes can be used in monitoring the biological systems/processes, or prognosis or diagnosis of diseases or disorders caused by deficiencies and/or imbalances of the analytes. For instances, homocysteine (Hcy), a thiolated amino acid; folic acid, an organic acid; and cholesterol, a lipid are all important prognostic and diagnostic markers for a wide range of cardiovascular diseases. Vitamins are important prognostic and diagnostic markers for various vitamin deficient diseases or disorders. Glucose, a monosaccharide, is a diagnostic marker for numerous glycemic conditions such as diabetic mellitus. Ethanol, an alcohol, is important in monitoring liquor consumption and potential liver damages. Bile acids or bile salts are important prognostic and diagnostic markers for certain cancers such as colon cancer. Monitoring uric acid is important because abnormally high concentration of uric acid is the diagnostic marker and cause of hyperuricemia leading to gout, which is very painful and can damage the kidney. In addition to these prognostic and diagnostic uses, methods for assaying analytes have applications in other agricultural, industrial or environmental protection processes where determining the presence, location and amount of the analytes is critical.
Assays for Homocysteine
Homocysteine (Hcy) is a thiol-containing amino acid formed from methionine during S-adenosylmethionine-dependent transmethylation reactions. Intracellular Hcy is remethylated to methionine, or is irreversibly catabolized in a series of reactions to form cysteine. Intracellular Hcy is exported into extracellular fluids such as blood and urine, and circulates mostly in oxidized form, and mainly bound to plasma protein (Refsum et al., Annu. Rev. Medicine, 49:31-62 (1998)). The amount of Hcy in plasma and urine reflects the balance between Hcy production and utilization. This balance may be perturbed by clinical states characterized by genetic disorders of enzymes involved in Hcy transsulfuration and remethylation (e.g., cystathionine xcex2-synthase and N5,10-methylenetetrahydrofolate reductase or dietary deficiency of vitamins (e.g., vitamin B6, B12 and folate) involved in Hcy metabolism (Baual, et al., Cleveland Clinic Journal of Medicine, 64:543-549 (1997)). In addition, plasma Hcy levels may also be perturbed by some medications such as anti-folate drugs (e.g., methotrexate) used for treatments of cancer or arthritis (Foody, et al., Clinician Reviews, 8:203-210 (1998)).
Severe cases of homocysteinemia are caused by homozygous defects in genes encoding for enzymes involved in Hcy metabolisms. In such cases, a defect in an enzyme involved in either Hcy remethylation or transsulfuration leads to as much as 50-fold elevations of Hcy in the blood and urine. The classic form of such a disorder, congenital homocystemia (Hcymia), is caused by homozygous defects in the gene encoding cystathionine xcex2-synthase (CBS). These individuals suffer from thromboembolic complications at an early age, which result in stroke, myocardial infarction, renovascular hypertension, intermittent claudication, mesenteric ischemic, and pulmonary embolism. Such patients may also exhibit mental retardation and other abnormalities resembling ectopia lensis and skeletal deformities (Perry T., Homocysteine: Selected aspects in Nyham W. L. ed. Hertable disorders of amino acid metabolism. New York, John Wiley and Sons, pp. 419-451 (1974)). It is also known that elevated Hcy levels in pregnant women is related to birth defects of children with neurotube closures (Scott et al., xe2x80x9cThe etiology of neural tube defectsxe2x80x9d in Graham, I., Refsum, H., Rosenberg, I. H., and Ureland P. M. ed. xe2x80x9cHomocysteine metabolism: from basic science to clinical medicinexe2x80x9d Kluwer Academic Publishers, Boston, pp. 133-136 (1995)). Thus, the diagnostic utility of Hcy determinations has been well documented in these clinical conditions.
It has been demonstrated that even mild or moderately elevated levels of Hcy also increase the risk of atherosclerosis of the coronary, cerebral and peripheral arteries and cardiovascular disease (Boushey, et al., JAMA, 274:1049-1057 (1995)). The prevalence of Hcymia was shown to be 42%, 28%, and 30% among patients with cerebral vascular disease, peripheral vascular disease and cardiovascular disease, respectively (Moghadasian, et al., Arch. Intern. Med., 157:2299-2307 (1997)). A meta-analysis of 27 clinical studies calculated that each increase of 5 xcexcM in Hcy level increases the risk for coronary artery disease by 60% in men and by 80% in women, which is equivalent to an increase of 20 mgxc2x7dlxe2x88x921 (0.5 mmolxc2x7dlxe2x88x921) in plasma cholesterol, suggesting that Hcy, as a risk factor, is as strong as cholesterol in general population. Results from these clinical studies concluded that hyperhomocysteinemia is an emerging new independent risk factor for cardiovascular disease, and may be accountable for half of all cardiovascular patients who do not have any of the established cardiovascular risk factors (e.g., hypertension, hypercholesterolemia, cigarette smoking, diabetes mellitus, marked obesity and physical activity).
Mild homocysteinemia is mainly caused by heterozygosity of enzyme defects. A common polymorphism in the gene for methylenetetrahydrofolate reductase appears to influence the sensitivity of homocysteine levels to folic acid deficiency (Boers, et al., J. Inher. Metab. Dis., 20:301-306 (1997)). Moreover, plasma homocysteine levels are also significantly increased in heart and renal transplant patients (Ueland, et al., J. Lab. Clin. Med., 114:473-501 (1989)), Alzheimer patients (Jacobsen, et al., Clin. Chem., 44:2238-2239 (1998)), as well as in patients of non-insulin-dependent diabetes mellitus (Ducloux, et al., Nephrol. Dial. Transplantl, 13:2890-2893 (1998)). The accumulating evidence linking elevated homocysteine with cardiovascular disease has prompted the initiation of double-blind, randomized and placebo controlled multicenter clinical trials to demonstrate the efficacy of lowering plasma Hcy in preventing or halting the progress of vascular disease (Diaz-Arrastia, et al., Arch. Neurol, 55:1407-1408 (1998)).
Determination of plasma homocysteine levels may become a common clinical practice in the near future. Today, cardiologists have already started to recommend their patients to examine their homocysteine levels especially for those who have family history in cardiovascular disease, or who have cardiovascular problem but with normal levels of cholesterol and other risk factors, and those who are older than 60 years-old.
The assay of total Hcy in plasma or serum is complicated by the fact that 70% of plasma Hcy is protein-bound, 20-30% exists as free symmetric or mostly asymmetric mixed disulfides, free reduced Hcy exists in only trace amounts (Stehouwer, et al., Kidney International, 55308-314 (1999)). As a risk factor for cardiovascular disease, the determination of total plasma Hcy levels (reduced, oxidized and protein-bound) has been recommended in clinical setting(Hornberger, et al., American J. of Public Health, 88:61-67 (1998)). Since 1982, several methods for determining total plasma Hcy have been described (Mansoor, et al., Anal. BioChem., 200:218-229 (1992); Steir, et al., Arch. Intern. Med., 158:1301-1306 (1998); Ueland, et al., Clin. Chem., 39:1764-1779 01993); and Ueland, et al., xe2x80x9cPlasma homocysteine and cardiovascular diseasexe2x80x9d in Francis, R. B. Jr. eds. Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function. New York, Marcel Dokker, pp. 183-236 (1992); see, also, Ueland, et al., xe2x80x9cPlasma homocysteine and cardiovascular diseasexe2x80x9d in Francis, R. B. Jr. eds. Atherosclerotic Cardiovascular Disease, Hemostasis, and Endothelial Function. New York, Marcel Dokker, pp. 183-236 (1992)). Most of these methods require sophisticated chromatographic techniques such as HPLC, capillary gas chromatography, or mass spectrometry (GC/MS) to directly or indirectly (e.g., enzymatic conversion of Hcy to SAH (S-adenosylhomocysteine) by SAH hydrolase followed by HPLC or TLC separation) measure Hcy. Radioenzymatic conversion of Hcy to radiolabelled SAH by SAH hydrolase prior to TLC separation has also been used. A feature common to all these methods includes the following four steps: (1) reduction of oxidized Hcy to reduced Hcy; (2) precolumn derivatization or enzymic conversion to SAH; (3) chromatographic separation; and (4) detection of the Hcy derivative or SAH. In these assays, chromatographic separation is the common key step of the prior art methods which are often time-consuming and cumbersome to perform. More particularly, these methods require highly specialized and sophisticated equipment and well-trained analytic specialists. The use of such equipment is generally not well accepted in routine clinical laboratory practice.
Immunoassays for Hcy that use a monoclonal antibody against SAH (Araki, et al., J. Chromatog., 422:43-52 (1987) are known. These assays are based upon conversion of Hcy to SAH, which is then detected by a monoclonal antibody. Monoclonal antibody against albumin-bound Hcy has been developed for determination of albumin-bound Hcy (Stabler, et al., J. Clin. Invest., 81:466-474 (1988)), which is the major fraction of total plasma Hcy. Other immmological protocols are also available (see, e.g., U.S. Pat. No. 5,885,767 and U.S. Pat. No. 5,631,127). Though immunoassays avoid a time-consuming chromatographic separation step and are amenable to automation, production of monoclonal antibody are expensive, somewhat unpredictable, and often require secondary or even tertiary antibodies for detection.
Despite the importance and wide applications of methods for assaying analytes, currently available methods for assaying analytes suffer from several deficiencies. First, for many analytes, specific binding partners are not readily available and this lack of specific binding partner often compromises the specificity of the assay method. Although such deficiency may be overcome by generating antibodies for macromolecule analytes, generating antibodies, especially monoclonal antibodies with the desired specificity and uniformity, is often time consuming and expensive. In addition, for many small molecule analytes, the option of generating antibodies is often not available because small molecules are poor antigens. Generation of antibodies against small molecules usually requires conjugation of the small molecules to macromolecules, and this often makes the antibody screening more tedious and laborious. Second, many methods for assaying analytes, especially small molecule analytes, involve chemical derivations and chromatographic separation can be time consuming. Third, many such assay methods use sophisticated and expensive analytical equipment such as HPLCs and GC/MS.
Therefore, it is an object herein to provide quick and simple and assays that address these deficiencies. It is also an object herein to provide such an assay for quantifying and/or detecting homocysteine in body fluids and body tissues.
Assays, particularly assays that are based on immunoassay formats, but that employ mutant analyte-binding enzymes that, substantially retain binding affinity or have enhanced binding affinity for desired analytes or an immediate analyte enzymatic conversion products but have attenuated catalytic activity, are provided. In place of antibodies, these assys use modified enzymes that retain binding affinity or having enhanced binding affinity, but have attenuated catalytic activity. These methods are designated substrate trapping methods; and the modified enzymes, are designated as xe2x80x9csubstrate trapping enzymes.xe2x80x9d The substrate trapping enzymes (also designated pseudoantibodies) and methods for preparing them are also provided. These substrate trapping enzymes are intended to replace antibodies, monoclonal, polyclonal or any mixture thereof, in reactions, methods, assays and processess in which an antibody (polyclonal, monoclonal or specific binding fragment thereof) is a reactant. They can also act as competitive inhibitors with analytes for binding to entities such as receptors and other anti-ligands and other analytes. Hence, they can be used in competitive binding assays in place of, for example, receptor agonists or modulators of receptor activity.
Any process or method, particularly immunoassays or assays in which an antibody aids in detection of a target analyte, can be modified as described herein, by substituting a substrate trapping enzyme for the antibody used in the process or method. The substrate trapping enzymes can be prepared by any method known to those of skill in the art by which the catalytic activity of an enzyme is substantially attenuated or eliminated, without affecting or without substantially reducing the binding affinity of the resulting modified enzyme for an analyte.
The methods are particularly useful for detecting analytes indicative of metabolic conditions, inborn errors of metabolism, such as hypothyroidism, galactosemia, phenylketonuria (PKU), and maple syrup urine disease; disease markers, such as glucose levels, cholesterol levels, Hcy levels and other such parameters in body fluid and tissue samples from mammals, including humans. The methods also include methods for detecting contanimants in food, for testing foods to quantitate certain nutrients, for screening blood. The assays readily can be automated.
Accordingly, methods in which an antibody is a reactant, wherein the improvement is replacement of the antibody with a substrate trapping enzymes as defined herein, are provided. The methods may also rely on competitive binding of the modified enzyme for a target analyte.
In another embodiment, a method is provided for assaying an analyte, preferably a small molecule analyte, in a sample by: a) contacting the sample with a mutant analyte-binding enzyme, the mutant enzyme substantially retains its binding affinity or has enhanced binding affinity for the analyte or an immediate analyte enzymatic conversion product but has attenuated catalytic activity; and b) detecting binding between the analyte or the immediate analyte enzymatic conversion product and the mutant analyte-binding enzyme.
The small molecule analyte to be assayed can be any analyte, including organic and inorganic molecules. Typically the small molecule to be assayed has a molecular weight that is about or less than 10,000 daltons. Preferably, the small molecule has a molecular weight that is about or less than 5,000 dalton.
Inorganic molecules include, but are not limited to, an inorganic ion such as a sodium, a potassium, a magnesium, a calcium, a chlorine, an iron, a copper, a zinc, a manganese, a cobalt, an iodine, a molybdenum, a vanadium, a nickel, a chromium, a fluorine, a silicon, a tin, a boron or an arsenic ion. Organic molecules include, but are not limited to, an amino acid, a peptide, typically containing less than about 10 amino acids, a nucleoside, a nucleotide, an oligonucleotide, typically containing less than about 10 nucleotides, a vitamin, a monosaccharide, an oligosaccharide containing less than 10 monosaccharides or a lipid.
The amino acids, include, but are not limited to, D- or L-amino-acids, including the building blocks of naturally-occurring peptides and protiens including Ala (A), Arg (R), Asn (N), Asp (D), Cys (C), Gln (Q), Glu (E), Gly (G), His (H), Ile (I), Leu (L), Lys (K), Met (M), Phe (F), Pro (P) Ser (S), Thr (T), Trp (W), Tyr (Y) and Val (V).
Nucleosides, include, but but are not limited to, adenosine, guanosine, cytidine, thymidine and uridine. Nucleotides include, but are not limited to, AMP, GMP, CMP, UMP, ADP, GDP, CDP, UDP, ATP, GTP, CTP, UTP, dAMP, dGMP, dCMP, dTMP, dADP, dGDP, dCDP, dTDP, dATP, dGTP, dCTP and dTTP.
Vitamins, include, but are not limited to, water-soluble vitamins such as thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxine, biotin, folate, vitamin B12 and ascorbic acid, fat-soluble vitamins such as vitamin A, vitamin D, vitamin E, and vitamin K.
Monosaccharides, include but are not limited to, D- or L-monosaccharides. Monosaccharides include, but are not limited to, triose, such as glyceraldehyde, tetroses such as erythrose and threose, pentoses such as ribose, arabinose, xylose, lyxose and ribulose, hexoses such as allose, altrose, glucose, mannose, gulose, idose, galactose, talose and fructose and heptose such as sedoheptulose.
Lipids, include, but are not limited to, triacylglycerols such as tristearin, tripalmitin and triolein, waxes, phosphoglycerides such as phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine, phosphatidylinositol and cardiolipin, sphingolipids such as sphingomyelin, cerebrosides and gangliosides, sterols such as cholesterol and stigmasterol and sterol fatty acid esters. The fatty acids can be saturated fatty acids such as lauric acid, myristic acid, palmitic acid, stearic acid, arachidic acid and lignoceric acid, or can be unsaturated fatty acids such as palmitoleic acid, oleic acid, linoleic acid, linolenic acid and arachidonic acid.
In an exemplary embodiment, mutant S-adenosylhomocysteine (SAH) hydrolases, substantially retaining binding affinity or having enhanced binding affinity for homocysteine (Hcy) or SAH but having attenuated catalytic activity, are provided. Also provided are methods, combinations, kits and articles of manufacture for assaying analytes, preferably small molecule analytes such as inorganic ions, amino acids (e.g., homocysteine), peptides, nucleosides, nucleotides, oligonucleotides, vitamins, monosaccharides (e.g., glucose), oligosaccharides, lipids (e.g., cholesterol), organic acids (e.g., folate species, bile acids and uric acids).
In another embodiment, provided herein are purified mutant SAH hydrolases, the mutant SAH hydrolases substantially retain their binding affinity or have enhanced binding affinity for homocysteine (Hcy) or SAH but have attenuated catalytic activity.
Examples of such mutant SAH hydrolases include those in which the the attenuated catalytic activity is caused by mutation(s) in the mutant SAH hydrolase""s binding site for NAD+, or mutation(s) in the mutant SAH hydrolase""s catalytic site or a combination thereof; those that have attenuated 5xe2x80x2-hydrolytic activity but substantially retain the 3xe2x80x2-oxidative activity; those that irreversibly bind SAH; those that have a Km for SAH that is about or less than 10.0 xcexcM; those that have a Kcat for SAH that is about or less than 0.1 Sxe2x88x921; those that have one or more insertional, deletional or point mutations; those that are derived from the sequence of amino acids set forth in SEQ ID No. 1 or encoded by the sequence of nucleotides set forth in SEQ ID No.2 and have one or, preferably at least two or more mutations selected from Phe302 to Ser (F302S), Lys186 to Ala (K186A), His301 to Asp (H301D), His353 to Ser (H353S), Arg343 to Ala (R343A), Asp190 to Ala (D190A), Phe82 to Ala (F82A), Thr157 to Leu (T157L), Cys195 to Asp (C195D), Asn181 to Asp (N181D), and deletion of Tyr432 (xcex94432); or those that are derived from the sequence of amino acids set forth in SEQ ID No. 1 or encoded by the sequence of nucleotides set forth in SEQ ID No. 2 and have a combination of Arg431 to Ala (R431A) and Lys426 to Arg (K426R) mutations; or any that hybridize under conditions of low, more preferably moderate, most preferably high, stringency along their full-length and have a Km at least about 10%, more preferably at least about 50% of the Km of the wildtype enzyme for the analyate or substrate, but having substantially attenuated catalytic activity to the coding portion of the sequence of nucleotides set forth in SEQ ID No. 1 or encoding the sequence of amino acids set forth in SEQ ID No. 2.
Isolated nucleic acid fragments encoding the above-described mutant SAH hydrolases, preferably in the form of plasmid or expression vectors, are also provided. Recombinant host cells, especially recombinant bacterial cells, yeast cells, fungal cells, plant cells, insect cells and animal cells, containing the plasmids or vectors are further provided. Methods for producing the mutant SAH hydrolases using the recombinant host cells are further provided.
Assays for Homocysteine and Metabolically Related Analytes
Assays for homocysteine, which as noted above, is a risk factor for cardiovascular disease and other diseases, are provided herein.
Homocysteine
In these embodiments, the small molecule to be assayed is homocysteine (Hcy) and the mutant analyte-binding enzymes are mutant Hcy-binding enzymes that substantially retain their binding affinity or that have enhanced binding affinity for Hcy or an immediate Hcy enzymatic conversion product but have attenuated catalytic activity.
Mutant Hcy-binding enzymes that can be used in the assay include those in which the attenuated catalytic activity is caused by mutation in the mutant enzyme""s binding site for its co-enzyme or for a non-Hcy substrate, or mutation in the mutant enzyme""s catalytic site or a combination thereof.
In another embodiment, the mutant enzyme is a mutant cystathionine xcex2-synthase and the attenuated catalytic activity is caused by mutation in the mutant cystathionine xcex2-synthase""s catalytic site, its binding site for pyridoxal 5xe2x80x2-phosphate or L-serine, or a combination thereof.
In another embodiment, the mutant enzyme is a mutant methionine synthase and the attenuated catalytic activity is caused by mutation in the mutant methionine synthase""s catalytic site, its binding site for vitamin B12 or 5-methyltetrahydrofolate (5-CH3-THF), or a combination thereof. More preferably, the mutant methionine synthase is an E. coli. methionine synthase, the mutant methionine synthase has one or more of the following mutations: His759Gly, Asp757Glu, Asp757Asn, and Ser801Ala.
In another embodiment, the mutant enzyme is a mutant methioninase and the attenuated catalytic activity is caused by mutation in the mutant methionine synthase""s catalytic site, its binding site for a compound with the formulae of Rxe2x80x2SH, in which Rxe2x80x2SH is a substituted thiol, where R is preferably alkyl, preferably lower alkyl (1 to 6 carbons, preferably 1 to 3 carbons, in a straight or branched chain), heteraryl, where the heteroatom is O, S or N, or aryl, which is substituted, such as with alkyl, preferably lower alkyl, or hal, or unsubstituted, preferably aryl or heteraryl with one ring or two to three fused rings, preferably with about 4 to 7 members in each ring, or combinations of any of the above.
In a preferred embodiment, the mutant enzyme is a mutant SAH hydrolase, where the mutant SAH hydrolase substantially retains its binding affinity or has enhanced binding affinity for Hcy or SAH but has attenuated catalytic activity. Examples of such mutant SAH hydrolases that can be used in the assay include those in which the attenuated catalytic activity is caused by mutation(s) in the mutant SAH hydrolase""s binding site for NAD+, or mutation(s) in the mutant SAH hydrolase""s catalytic site or a combination thereof; those that have attenuated 5xe2x80x2-hydrolytic activity but substantially retains its 3xe2x80x2-oxidative activity; those that irreversibly bind SAH; those that have a Km for SAH that is about or less than 10.0 xcexcM; those that have a Kcat for SAH that is about or less than 0.1 Sxe2x88x921; those that have one or more insertional, deletional or point mutation; those that are derived from the sequence of amino acids set forth in SEQ ID No. 1 or encoded by the sequence of nucleotides set forth in SEQ ID No. 2 but have one or more of the following mutations: Phe302 to Ser (F302S), Lys186 to Ala (K186A), His301 to Asp (H301D), His353 to Ser (H353S), Arg343 to Ala (R343A), Asp190 to Ala (D190A), Phe82 to Ala (F82A), Thr157 to Leu (T157L), Cys195 to Asp (C195D), Asn181 to Asp (N181D), and deletion of Tyr432 (xcex94432); or those that are derived from a sequence of amino acids set forth in SEQ ID No. 1 or encoded by the sequence of nucleotides set forth in SEQ ID No. 2 and have a combination of Arg431 to Ala (R431A) and Lys426 to Arg (K426R) mutations or any that hybridize under conditions of low, more preferably moderate, most preferably high, stringency along their full-length and have a Km at least about 10%, more preferably at least about 50% of the Km of the wildtype enzyme for the analyate or substrate, but having substantially attenuated catalytic activity.
In one embodiment that uses a mutant SAH hydrolase, oxidized Hcy in the sample is converted into reduced Hcy prior to the contact between the sample and the mutant SAH hydrolase. The oxidized Hcy in the sample is converted into reduced Hcy by a reducing agent, such as, but are not limited to, tri-n-butyphosphine (TBP), xcex2-ME, DTT, dithioerythritol, thioglycolic acid, glutathione, tris(2-carbxyethyl)phosphine, sodium cyanoborohydride, NaBH4, KBH4 and free metals.
In another embodiment that uses a mutant SAH hydrolase, prior to the contact between the sample and the mutant SAH hydrolase, the Hcy in the sample is converted into SAH. More preferably, the Hcy in the sample is converted into SAH by a wild-type SAH hydrolase. Also more preferably, the SAH in the sample is contacted with the mutant SAH hydrolase in the presence of a SAH hydrolase catalysis inhibitor, such as, but are not limited to, neplanocin A or thimersol.
In another embodiment that uses a mutant SAH hydrolase, prior to the contact between the SAH and the mutant SAH hydrolase, free adenosine is removed or degraded. More preferably, free adenosine is degraded by combined effect of adenosine deaminase, purine nucleoside phosphorylase and xanthine oxidase.
In another embodiment that uses a mutant SAH hydrolase, the SAH is contacted with the mutant SAH hydrolase in the presence of a labelled SAH or a derivative or an analog thereof, whereby the amount of the labeled SAH bound to the mutant SAH hydrolase inversely relates to amount of the SAH in the sample. More preferably, the labelled SAH derivative or analog is a fluorescence labelled adenosyl-cysteine.
In another embodiment that uses a mutant SAH hydrolase, the mutant SAH hydrolase is labelled mutant SAH hydrolase. More preferably, the mutant SAH hydrolase is labelled by fluorescence.
In still another embodiment, the mutant enzyme is a mutant betaine-homocysteine methyltransferase and the attenuated catalytic activity is caused by mutation in the mutant betaine-homocysteine methyltransferase""s binding site for betaine, its catalytic site, or a combination thereof.
In another embodiment, the Hcy assay is performed in combination with assays for other analytes associated with cardiovasicular disease and/or regulation of Hcy levels, such as assays for cholesterol and/or folic acid.
Folate
In another embodiment, the mutant enzyme is a mutant methionine synthase. In this embodiment, the folate species can be a 5,-methyltetrahydrofolate, the mutant folate-species-binding enzyme is a mutant methionine synthase, and the attenuated catalytic activity of the mutant methionine synthase is caused by mutation in its catalytic site, its binding site for vitamin B12, Hcy, or a combination thereof.
In another embodiment, the folate species is tetrahydrofolate, the mutant folate-species-binding enzyme is a mutant tetrahydrofolate methyltransferase, and the attenuated catalytic activity of the mutant tetrahydrofolate methyltransferase is caused by mutation in its catalytic site, its binding site for serine, or a combination thereof.
In still another embodiment, the folate species is 5,10,-methylene tetrahydrofolate, the mutant folate-species-binding enzyme is a mutant methylenetetrahydrofolate reductase, and the attenuated catalytic activity of the methylenetetrahydrofolate reductase is caused by mutation in its catalytic site.
In yet another embodiment, the folate species is 5,10,-methylene tetrahydrofolate, the mutant folate-species-binding enzyme is a mutant folypolyglutamate synthase, and the attenuated catalytic activity of the folypolyglutamate synthase is caused by mutation in its catalytic site, its binding site for ATP, L-glutamate, Mg2+, or a combination thereof. In yet another preferred embodiment, the folate species is dihydrofolate, the mutant folate-species-binding enzyme is a mutant dihydrofolate reductase, and the attenuated catalytic activity of the mutant dihydrofolate reductase is caused by mutation in its catalytic site, its binding site for NADPH, or a combination thereof. More preferably, the mutant dihydrofolate reductase is a Lactobacillus casei dihydrofolate reductase having the Arg43Ala or Trp21His mutation (Basran et al., Protein Eng., 10(7):815-26 91997)).
In yet another embodiment, the folate species is 5,10,-methylene tetrahydrofolate (5,10-methylene-FH4), the mutant folate-species-binding enzyme is a mutant thymidylate synthase, and the attenuated catalytic activity of the mutant thymidylate synthase is caused by mutation in its catalytic site, its binding site for dUMP, or a combination thereof. More preferably, the mutant thymidylate synthase is a human thymidylate synthase having a mutation selected from of Tyr6His, Glu214Ser, Ser216Ala, Ser216Leu, Asn229Ala and His199X, where X is any amino acid that is not His (Schiffer et al., Biochemistry, 34(50):16279-87 (1995); Steadman et al., Biochemistry, 37:7089-7095 (1998); Williams et al., Biochemistry, 37(20):7096-102 (1998); Finer-Moore et al., J. Mol. Biol., 276(1):113-29 (1998); and Finer-Moore et al., Biochemistry, 35(16):5125-36 (1996)). Also more preferably, the mutant thymidylate synthase is an E. coli thymidylate synthase having an Arg126Glu mutation (Strop et al., Protein Sci., 6(12):2504-11 (1997)) or a Lactobacillus casei thymidylate synthase having a V316Am mutation (Carreras et al., Biochemistry, 31 (26):6038-44 (1992)).
Cholesterol
In another embodiment, the analyte is cholesterol and the mutant analyte-binding enzyme is a mutant cholesterol-binding enzyme, where the mutant enzyme substantially retains its binding affinity or has enhanced binding affinity for cholesterol but has attenuated catalytic activity. In a preferred embodiment, the mutant cholesterol-binding enzyme is a mutant cholesterol esterase, and the attenuated catalytic activity of the mutant cholesterol esterase is caused by mutation in its catalytic site, its binding site for H2O or a combination thereof. More preferably, the cholesterol esterase is a pancreatic cholesterol esterase having a Ser194Thr or Ser194Ala mutation (DiPersio et al., J. Biol. Chem., 265(28):16801-6 (1990)). In another preferred embodiment, the mutant cholesterol-binding enzyme is a mutant cholesterol oxidase, and the attenuated catalytic activity of the mutant cholesterol oxidase is caused by mutation in its catalytic site, its binding site for O2 or a combination thereof. More preferably, the cholesterol oxidase is a Brevibacterium sterolicum cholesterol oxidase having a His447Asn or His447Gln mutation (Yue et al., Biochemistry, 38(14):4277-86 (1999)).
Bile Acid (salt)
In still another specific embodiment, the small molecule analyte is a bile acid (salt) and the mutant analyte-binding enzyme is a mutant bile-acid (salt)-binding enzyme, the mutant enzyme substantially retains its binding affinity or has enhanced binding affinity for the bile acid (salt) but has attenuated catalytic activity. Preferably, the mutant bile-acid (salt)-binding enzyme is a mutant 3-xcex1-hydroxy steroid dehydrogenase, and the attenuated catalytic activity of the mutant 3-xcex1-hydroxy steroid dehydrogenase is caused by mutation in its catalytic site, its binding site for NAD+ or a combination thereof.
Assays for Disorders Associated with Glucose Metabolism
In yet another specific embodiment, the small molecule analyte is glucose and the mutant analyte-binding enzyme is a mutant glucose-binding enzyme, the mutant enzyme substantially retains its binding affinity or has enhanced binding affinity for glucose but has attenuated catalytic activity. Preferably, the mutant glucose-binding enzyme is a Clostridium thermosulfurogenes glucose isomerase having a mutation selected from His101Phe, His101Glu, His101Gln, His101Asp and His101Asn (Lee et al., J. Biol. Chem., 265(31):19082-90 (1990)). Also preferably, the mutant glucose-binding enzyme is a mutant hexokinase or glucokinase, and the attenuated catalytic activity of the mutant hexokinase or glucokinase is caused by mutation in its catalytic site, its binding site for ATP or Mg2+, or a combination thereof. Further preferably, the mutant glucose-binding enzyme is a mutant glucose oxidase, and the attenuated catalytic activity of the mutant glucose oxidase is caused by mutation in its catalytic site, its binding site for H2O or O2, or a combination thereof. Any disorders associated with glucose metabolism may be monitored or assessed.
Ethanol
In yet another specific embodiment, the small molecule analyte is ethanol and the mutant analyte-binding enzyme is a mutant ethanol-binding enzyme, the mutant enzyme substantially retains its binding affinity or has enhanced binding affinity for ethanol but has attenuated catalytic activity. Preferably, the mutant ethanol-binding enzyme is a mutant alcohol dehydrogenase, and the attenuated catalytic activity of the mutant alcohol dehydrogenase is caused by mutation in its catalytic site, its binding site for NAD+ or Zn2+, or a combination thereof. More preferably, the mutant alcohol dehydrogenase is a human liver alcohol dehydrogenase having a His51Gln mutation (Ehrig et al., Biochemistry, 30(4):1062-8 (1991)). Also more preferably, the mutant alcohol dehydrogenase is a horse liver alcohol dehydrogenase having a Phe93Trp or Val203Ala mutation (Bahnson et al., Proc. Natl. Acad. Sci., 94(24):12797-802 (1997); Colby et al., Biochemistry, 37(26):9295-304 (1998)).
Assays for Disorders, Such as Gout, Associated with Uric Acid Acid Metabolism
In another exemplary embodiment, the small molecule analyte is uric acid and the mutant analyte-binding enzyme is a mutant uric-acid-binding enzyme, the mutant enzyme substantially retains its binding affinity or has enhanced binding affinity for uric acid but has attenuated catalytic activity. Preferably, the mutant uric-acid-binding enzyme is a mutant urate oxidase, and the attenuated catalytic activity of the mutant urate oxidase is caused by mutation in its catalytic site, its binding site for O2, H2O, or copper ion, or a combination thereof. More preferably, the mutant urate oxidase is a rat urate oxidase having a mutation selected from H127Y, H129Y and F131S (Chu et al., Ann. N.Y. Acad. Sci., 804:781-6 (1996)).
In all embodiments, the sample being assayed typically is a body fluid or tissue, including, but are not limited to blood, urine, cerebral spinal fluid, synovial fluid, amniotic fluid, and tissue samples, such as biopsied tissues. Preferably, the body fluid is blood or urine. More preferably, the blood sample is further separated into a plasma or sera fraction.
Further provided herein are combinations that include: a) a mutant analyte-binding enzyme, the mutant enzyme substantially retains its binding affinity or has enhanced binding affinity for the analyte or an immediate analyte enzymatic conversion product but has attenuated catalytic activity; and b) reagents and or other means for detecting binding between the analyte or the immediate analyte enzymatic conversion product with the mutant analyte-binding enzyme. Preferably, binding between the analyte or the immediate analyte enzymatic conversion product with the mutant analyte-binding enzyme is detected using a labelled analyte, a labelled immediate analyte enzymatic conversion product, or a derivative or an analog thereof, or a labelled mutant analyte-binding enzyme. Also preferably, the combination where the analyte is Hcy further also includes reagents for detecting cholesterol and/or folic acid.
Finally, kits and articles of manufacture that include the above combinations and optionally instructions for performing the assay of interest are provided. Articles of manufacture that contain the mutant enzymes with a label indicating the assay in which the enzyme is used, and also packaging material that contains the enzyme.
Particular compositions, combinations, kits and articles of manufacture for assaying analytes, preferably small molecule analytes, are described in the sections and subsections that follow.